HPLC and FPLC

1. HPLC and FPLC
HPLC and FPLC
By: Britney Sykes

2.  Several chromatographic techniques
 Even though each method utilizes different
techniques to separate compounds, the
principles are the same.
 Common to all:
 Stationary phase-
 a solid or a liquid supported on a solid
 Mobile phase-
 A liquid or gas
Chromatography Theory Review

3.  As the mobile phases passes through the
stationary phase, it carries the components of
the sample mixture with it.
 The components of the sample will be attracted to
the stationary phase, but there will also be a
competing attraction for the mobile phase.
 Each component will have its own characteristic
balance of attraction to the mobile/stationary
phase.
 So the components will not move at the same
speed and are separated.
Chromatography Theory Review

4. Column Chromatography
 Similar to thin layer
chromatography
 Stationary phase =
silica gel on support
 Mobile phase = liquid solvent
 In column chromatography,
this stationary phase is
packed into a vertical glass
column.
 Mobile phase moves down
the column as a result of
gravity.

5. Column Chromatography
 Blue compound = more polar
 Adsorb more to the
silica gel
 Elutes slower
 Yellow compound =
less polar
 Spends much of its
time in the mobile
phase
 Elutes faster
 Example of column chromatography separation:

6. HPLC Introduction:
 HPLC = improved form of column chromatography
 Instead of the mobile phase moving through the
column as a result of gravity, it is forced through
the column under high pressure.
 Typical operating pressures: 500-6000psi
 To get improved separation – smaller sized
packing material is required (<10µm).
 Smaller packing = greater resistance to flow
 Low flow rate = solute diffusion
 Higher pressures needed to generate the needed solvent flow
 Gravity is too slow- high pressure greatly speeds up the
procedure.

7. 1903: Russian botanist Mikhail Tswett
 Separated plant pigments through
column adsorption chromatography
 Packed open glass column with particles
 Calcium carbonate and alumina
 Poured sample into column, along with
pure solvent
 As the sample moved down the vertical
column, different colored bands could
be seen.
 Bands correlated to the sample
components.
 Coined the term chromatography from
the Latin word meaning “color writing”.
HPLC History

8. Early 1950s: First appearance of GC
 Almost immediately became popular.
 Work began on improving LC
1964: J. Calvin Giddings
 Published a paper entitled “Comparison of the
Theoretical Limit of Separating Ability in Gas and
Liquid Chromatography” in the journal Analytical
Chemistry.
 Outlined ways to improve LC: smaller packing size,
increased pressure
 In theory, he demonstrated how LC could actually be more
efficient than GC.
 Increased number of theoretical plates
HPLC History

9. HPLC History
1966: Horváth
 Built the first HPLC instrument and gave it its name
 HPLC = High Pressure Liquid Chromatography.
1970’s: HPLC became popular with an increase in
technology
 Improved columns and detectors
 Production of small silica packing material
 By 1972 particle sizes less than 10µm were introduced
 This allowed for more precise and rapid separations.
 As new technology continued to develop, HPLC
became more efficient.
 HPLC = High Performance Liquid Chromatography

10. Overview of the HPLC Process
 Mobile phase pumped through column at high pressure.
 Sample is injected into the system.
 Separation occurs as the mobile phase and sample are
pumped through the column.
 Each sample component will elute from the column, one at a
time, and will be detected by one of several possible
detector types.
 The response of the detector to each component eluted will
be displayed on a chart or computer screen.
 Known as a chromatogram.
 Each compound eluted will show up as a peak on this chromatogram.
 Data processing equipment are used to analyze the data
generated.
http://www.waters.com/WatersDivision/flash/hplc_primer/hplcsys_primer.html

11. Common Reverse Phase
Solvents
 Methanol CH3OH
• Acetonitrile CH3CN
• Tetrahydrofuran
• Water H2O

12. Bonded Phases
 C-2 Ethyl Silyl -Si-CH2-CH3
• CN Cyanopropyl Silyl -Si-(CH2)3-CN
• C-18 Octadecyl Silane -Si-(CH2)17-CH3
• C-8 Octyl Silyl -Si-(CH2)7-CH3

13. Diagram of HPLC Apparatus:
1.
http://www.cem.msu.edu/~cem333/Week16.pdf

14. Design & Operation of an HPLC
Instrument
1) Mobile phase degassing:
 Dissolved gases in the mobile phase can come out of
solution and form bubbles as the pressure changes
from the column entrance to the exit.
 May block flow through the system
 Sparging is used to remove any dissolved gas from
the mobile phase.
 An inert and virtually insoluble gas, such as helium, is forced
into the mobile phase solution and drives out any dissolved
gas.
 Degassing may also be achieved by filtering the
mobile phase under a vacuum.

15. Design & Operation of an HPLC
Instrument
2) Solvent reservoirs:
 Individual reservoirs store the mobile phase
components until
they are mixed
and used.
 May also manually
prepare the mobile
phase mixture and
store in a single
reservoir.
2.

16. 3) Mobile phase mixing:
 Solvent proportioning valve can be programmed
to mix specific
amounts of solvent
from the various
reservoirs to
produce the
desired mobile
phase composition.
Design & Operation of an HPLC
Instrument
3.

17. 3) Mobile phase mixing:
 Isocratic elution:
 operate at a single, constant mobile phase composition
 Gradient elution:
 Vary the mobile phase composition with time
 If there is a wide polarity range of components to be eluted.
 Allows for faster runs.
 Ex: mobile phase composition can be programmed to vary
from 75% water: 25% acetonitrile at time zero to
25% water: 75% acetonitrile at the end of the run.
 More polar components will tend to elute first.
 More non-polar components will elute later in the gradient.
Design & Operation of an HPLC
Instrument

18. Design & Operation of an HPLC
Instrument
4) HPLC pump:
 Fill stroke: mobile phase is pulled from the
solvent side
 Exhaust stroke:
the mobile phase
is pushed from the
injector to the
column head.
 This is where the
high pressure is
generated
4.

19. 4) HPLC pump:
 Most common =
reciprocating piston type
 Flow rates change during
pumping cycle
 Want to minimize flow surges
 Pulse dampener
 Dual pistons
 While one piston fills, the other delivers
Design & Operation of an HPLC
Instrument
http://www.lcresources.com/resources/getstart/2b01.htm

20. 5) Injector:
 Introduces the sample into the mobile phase stream to
be carried into the
column.
 Syringe = impractical
for use in highly
pressurized systems.
 Rotary injection
valve is used.
 For more
information visit:
http://www.lcresources.com/resources/getstart/2c01.htm
Design & Operation of an HPLC
Instrument
5.

21. 6) Column:
 Usually constructed of stainless steel
 glass or Tygon may
be used for lower
pressure applications
(<600 psi).
 Length: 5-100cm
 10 to 20cm common
 Diameter:
 Typical: 2.1, 3.2, or
4.5mm
 Up to 30mm for preparative applications
Design & Operation of an HPLC
Instrument
6.

22.  Column packing:
 Usually spherical silica particles of
uniform diameter (2-10µm)
 The smaller particles yield higher
separation efficiencies.
 The silica particles are very porous
 Allows for greater surface area for
interactions between the stationary
phase and the analytes.
 Other packing materials may also be
used:
 Zirconia (ZrO2)
http://www.lcresources.com/resources/getstart/3a01.htm
Design & Operation of an HPLC
Instrument
http://hplc.chem.shu.edu/NEW/HP
LC_Book/Adsorbents/ads_part.html

23. 6) Column:
 Guard column: Protects the analytical column
 Particles
 Interferences
 Prolongs the life of the analytical column
 Analytical column: Performs the separation
Design & Operation of an HPLC
Instrument

24. 7) Detector:
 The component that
emits a response due
to the eluting sample
compound and
subsequently signals
a peak on the
chromatogram.
 A wide variety of
detectors exist.
 Must have high sensitivity- small sample sizes are used with
most HPLC columns
Design & Operation of an HPLC
Instrument
7.

25. Detection in HPLC
*There are six major HPLC detectors:
 Refractive Index (RI) Detector
 Evaporative Light Scattering Detector (ELSD)
 UV/VIS Absorption Detectors
 The Fluorescence Detector
 Electrochemical Detectors (ECDs)
 Conductivity Detector
* The type of detector
utilized depends on
the characteristics
of the analyte of
interest. http://www.waters.com/WatersDivision/Contentd.asp?watersit=JDRS-6UXGZ4

26. Refractive Index Detector
 Based on the principle that every transparent
substance will slow the speed of light passing through
it.
 Results in the bending of light
as it passes to another material
of different density.
 Refractive index = how much
the light is bent
 The presence of analyte molecules
in the mobile phase will generally
change its RI by an amount
almost linearly proportional to its concentrations.
http://farside.ph.utexas.edu/teaching/302l/
lectures/img1154.png

27. Refractive Index Detector
 Affected by slight changes in mobile phase
composition and temperature.
 Universal-based on a property of the mobile phase
 It is used for analytes which give no response with
other more sensitive and selective detectors.
 RI = general
 responds to the presence of all solutes in the mobile
phase.
 Reference= mobile phase
 Sample= column effluent
 Detector measures the
differences between the RI of
the reference and the sample. http://hplc.chem.shu.edu/HPLC/index.html

28.  Analyte particles don’t scatter light
when dissolved in a liquid mobile
phase.
 Three steps:
1) Nebulize the mobile phase effuent into
droplets.
 Passes through a needle and mixes
with hydrogen gas.
2) Evaporate each of these droplets.
 Leaves behind a small particle of
nonvolatile analyte
3) Light scattering
 Sample particles pass through a cell
and scatter light from a laser beam
which is detected and generates a
signal.
Evaporative Light Scattering Detector
(ELSD)
http://www.sedere.com/WLD/whatis.html

29. UV/VIS Absorption Detectors
 Different compounds will absorb different amounts of
light in the UV and visible regions.
 A beam of UV light is shined through the analyte after
it is eluted from the column.
 A detector is positioned on the opposite side which
can measure how much light is absorbed and
transmitted.
 The amount of light
absorbed will depend on
the amount of the
compound that is passing
through the beam. http://www.chemguide.co.uk/analysis/chromatography/hplc.html

30. UV/VIS Absorption Detectors
 Beer-Lambert law: A=εbc
 absorbance is proportional to the compound
concentration.
 Fixed Wavelength: measures at one wavelength,
usually 254 nm
 Variable Wavelength: measures at one wavelength
at a time, but can detect over a wide range of
wavelengths
 Diode Array Detector (DAD): measures a spectrum
of wavelengths simultaneously

31. The Fluorescence Detector
 Measure the ability of a compound to absorb then
re-emit light at given wavelengths
 Some compounds will absorb
specific wavelengths of light which,
raising it to a higher energy state.
 When the compound returns to its
ground state, it will release a
specific wavelength of light which
can be detected.
 Not all compounds can fluoresce /
more selective than UV/VIS detection.
http://mekentosj.com/science/fret/images/
fluorescence.jpg

32. Electrochemical Detectors (ECDs):
 Electrochemical Detectors (ECDs):
 Used for compounds that undergo
oxidation/reduction reactions.
 Detector measures the current resulting
from an oxidation/reduction reaction of
the analyte at a suitable electrode.
 Current level is directly proportional to
the concentration of analyte present.
 Conductivity Detector:
 Records how the mobile phase
conductivity changes as different sample
components are eluted from the column.
http://hplc.chem.shu.edu/HPLC/index.html
http://hplc.chem.shu.edu/HPLC/index.html

33. Interfacing HPLC to Mass Spectrometry
 Mass Spectrometry = an analytical tool used
to measure the molecular mass of a sample.
 Measures the mass to
charge ratio
 Allows for the definitive
identification of each
sample component.
 Most selective HPLC
detector, but also the
most expensive.
http://www.chem.queensu.ca/FACILI
TIES/NMR/nmr/mass-spec/index.htm

34. Picture of a Typical HPLC System
http://www.waters.com/WatersDivision/ContentD.asp?watersit=JDRS-6UXGYA&WT.svl=1

35. Retention Time- tR
 The elapsed time between the time of analyte
injection and the time which the maximum peak
height for that compound is detected.
 Different compounds will have different retention
times.
 Each compound will have its own characteristic balance
of attraction to the mobile/stationary phase.
 So they will not move at the same speed.
 Running conditions can also effect tR:
 Pressure used, nature of the stationary phase, mobile
phase composition, temperature of the column

36. Retention Time- tR
 If you are careful to keep the conditions
constant, you may use tR to help you identify
compounds present.
 Must have measured tR for the pure compounds
under identical conditions.

37. Determining Concentration
 In most cases, sample peaks on the chromatogram
can be used to
estimate the
amount of a
compound
present.
 The more
concentrated,
the stronger
the signal, the
larger the
peak. http://www.waters.com/WatersDivision/Contentd.asp?watersit=JDRS-6UXGZ4

38.  tR: Retention time
 t’R: Adjusted retention time = (tR- Tm)
 Tm: Dead time
 W0,5: Peak width at half height
 h: Height of signal

39. Types of HPLC
 There are numerous types of HPLC which
vary in their separation chemistry.
 All chromatographic modes are possible:
 Ion-exchange
 Size exclusion
 Also can vary the stationary & mobile
phases:
 Normal phase HPLC
 Reverse phase HPLC

40. Chromatographic Modes of HPLC
 Ion exchange:
 Used with ionic or ionizable samples.
 Stationary phase has a charged surface.
 opposite charge to the sample ions
 The mobile phase = aqueous buffer
 The stronger the charge on the analyte, the more it will be
attracted to the stationary phase, the slower it will elute.
 Size exclusion:
 Sample separated based on size.
 Stationary phase has specific pore sizes.
 Larger molecules elute quickly.
 Smaller molecules penetrate inside the pores of the
stationary phase and elute later.

41. Normal Phase HPLC
 Stationary phase: polar, silica particles
 Mobile phase: non-polar solvent or mixture of
solvents
 Polar compounds:
 Will have a higher affinity for the polar,
stationary phase
 Will elute slower
 Non-polar compounds:
 Will have a higher affinity for the non-polar,
mobile phase
 Will elute faster

42. Reverse Phase HPLC
 Stationary phase: non-polar
 Non-polar organic groups are covalently attached to the
silica stationary particles.
 Most common attachment is a long-chain
n-C18 hydrocarbon
 Octadecyl silyl group, ODS
 Mobile phase: polar liquid or
mixture of liquids
 Polar analytes will spend more time
in the polar mobile phase.
 Will elute quicker than non-polar analytes
 Most common type of HPLC used today.
http://www.lcresources.com/
resources/getstart/3a01.htm

43. What is Ultra Performance Liquid
Chromatography?
 2004: Further advances in column technology
and chromatography instrumentation
 Utilized even smaller packing particle sizes (1.7µm)
 Higher pressures (15000psi)
 Allowed for significant increases in LC speed,
reproducibility, and sensitivity.
 New research utilizing particle sizes as small as
1µm and pressures up to 100,000psi!
WHO KNOWS WHAT THE FUTURE MAY BRING!

44. HPLC Applications
 Can be used to isolated and purify compounds
for further use.
 Can be used to identify the presence of specific
compounds in a sample.
 Can be used to determine the concentration of
a specific compound in a sample.
 Can be used to perform chemical separations
 Enantiomers
 Biomolecules

45. HPLC Applications
*HPLC has an vast amount of current & future applications*
 Some uses include:
 Forensics: analysis of explosives, drugs, fibers, etc.
 Proteomics: can be used to separate and purify protein
samples
 Can separate & purify other biomolecules such as: carbohydrates,
lipids, nucleic acids, pigments, proteins, steroids
 Study of disease: can be used to measure the presence &
abundance of specific biomolecules correlating to disease
manifestation.
 Pharmaceutical Research: all areas including early
identification of clinically relevant molecules to large-scale
processing and purification.

46. FPLC- A Modification of HPLC
 In 1982 Pharmacia introduced a new
chromatographic method called FPLC.
 FPLC = Fast Protein Liquid Chromatography
 FPLC is basically a “protein friendly” HPLC system.
 Stainless steel components replaced with glass and
plastic.
 Stainless steel was thought to denature proteins
 Also many ion-exchange separations of proteins involve salt
gradients; thought that these conditions could results in
attack of stainless steel systems.
 FPLC can also be used to separate other biologically
active molecules, such as nucleic acid.

47. FPLC- A Modification of HPLC
 FPLC is an intermediate between classical
column chromatography and HPLC.
 FPLC pump delivers a solvent flow rate in the
range of 1-499ml/hr
 HPLC pump= 0.010-10ml/min
 FPLC operating pressure: 0-40 bar
 HPLC= 1-400bar
 classic chromatography= atmospheric pressure
 Since lower pressures are used in FPLC than in
HPLC, a wider range of column supports are
possible.

48. Applications of FPLC/HPLC to Proteomics
 Protein characterization and measurement is
essential to understanding life at the
molecular level.
 The first step in protein analysis is isolation
and purification.
 Proteins can be separated by FPLC/HPLC in
various forms.
 Reversed phase
 Ion exchange
 Size exclusion
 Affinity
Used to isolate/purify without loss of
biological activity

49. Applications of FPLC/HPLC to Proteomics
 Not only can FPLC/HPLC systems be used to
isolate and purify proteins, but they can be
coupled to other instruments for further
analysis.
 UV/VIS
 Mass Spectrometer
 This instrument interfacing can allow for the
determination of:
 Protein amino acid sequence
 Structural information
 Functional information

50. http://www.msu.edu/~gallego7/MassSpect/MSandPMM.htm

51. The Impact of HPLC/FPLC
 HPLC/FPLC has such widespread application it
is impossible to convey its extensive impact.
 Has many advantages in situations were a
nonvolatile or thermally unstable sample must be
separated.
 As with many biochemical samples
 Great speed and resolution
 Resolution = how well solutes are separated
 Columns don’t have to be repacked
 Adaptable to large-scale, preparative procedures.

52. References
 Ashcroft, A.E. An Introduction to Mass Spectrometry. URL:
http://www.astbury.leeds.ac.uk/facil/MStut/mstutorial.htm
. Accessed: July 1, 2007.
 Detectors and Detection Limits. URL: http://kerouac.pharm
.uky.edu/ASRG/HPLC/detectors.html. Accessed: July 2,
2007.
 Filmore, David; Lesney, Mark S. Performing Under
Pressure: The Rise of HPLC. URL: http://pubs.acs.org/jour
nals/chromatography/chap4.html. Accessed: July 1, 2007.
 FPLC??. URL: http://www.lcresources.com/discus/messag
es/5133/2395.html?TuesdayAugust520031156am. Accesed
July2, 2007.
 Getting Started in HPLC. URL: http://www.lcresources.co
m/resources/get start/. Accessed: June 22, 2007.

53. References:
 High Performance Liquid Chromatography- HPLC. URL:
http://www.chemguide.co.uk/analysis/chromatography/hplc.
html#top. Accessed: June 15, 2007.
 High Performance Liquid Chromatography (HPLC)
Primer.URL: http://www.waters.com/WatersDivision/Conten
td.asp?Watersit =JDRS-6UXGZ4. Accessed: June 22, 2007.
 Kazakevich, Y; McNair, H.M. Basic Liquid Chromatography.
URL: http://hplc.chem.shu.edu/HPLC/index.html. Accessed:
June 22, 2007.
 Boyer, Rodney. Modern Experimental Biochemistry.3rd Ed.
Addison Wesley Longman. San Frisco, CA. 2000. 87-100.
 Robertson, James W.; Frame, Eileen M.; Frame, George M.
Undergraduate Instrumental Analysis. Marcel Dekker: New
York, NY. 2005.797-836.